Gene Cloning

What Does It Mean: “To Clone”?What Does It Mean: “To Clone”?

Clone: a collection of molecules or cells, all identical to an original molecule or cell

To "clone a gene" is to make many copies of it - for example, by

replicating it in a culture of bacteria.Cloned gene can be a normal copy of a gene (= “wild type”).Cloned gene can be an altered version of a gene (= “mutant”).Recombinant DNA technology makes manipulating genes

possible.

02/20/15Asheesh Kumar Pandey ([email protected])

One basic cloning technique begins with the insertion of a foreign gene into a bacterial plasmid.

02/20/15Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

Fig. 20.1 Asheesh Kumar Pandey ([email protected])

The objectives of Recombinant DNA technology include: ◦ Identifying genes◦ Isolating genes◦ Modifying genes◦ Re-expressing genes in other hosts or

organisms




Genetic engineering produces proteins that offer advantages over proteins isolated from other biological sources. These advantages include:◦ High purity

◦ High specific activity

◦ Steady supply

◦ Batch-to-batch consistency


Case Study: The Use of Recombinant Case Study: The Use of Recombinant DNA to Produce Human InsulinDNA to Produce Human Insulin


Why synthesize human insulin?Why synthesize human insulin?Patients’ immune systems do not

produce antibodies against human insulin as they do with bovine or porcine insulin

Projected decline in the production of animal-derived insulin

Need for a more reliable and sustainable method of obtaining the product


Why is insulin needed?Why is insulin needed?Protein hormone produced by beta cells

of islets of Langerhans in the pancreasRegulates blood sugar by allowing uptake

of glucose from bloodstream into body cells

Patients with diabetes have insufficient or impaired production of insulin


Structure of InsulinStructure of InsulinTwo polypeptide chains; one with 21

amino acids and the second with 30 amino acids

Chains are linked via a disulfide bond

Gene encoding the insulin protein is found on chromosome 11


Recombinant DNA TechniqueRecombinant DNA Technique

Restriction enzymes used to cut out insulin gene and to cut a bacterial

(E. coli) plasmid at the same “sticky ends”


Recombinant DNA TechniqueRecombinant DNA TechniqueMutant strains of E. coli used to avoid

bacteria attacking “foreign” genesInsert insulin gene next to E. coli

B-galactosidase gene which controls transcription

Bacterial cells replicate and make copies of insulin gene


Recombinant DNA TechniqueRecombinant DNA TechniqueInsulin protein is purified (B-galactosidase

removed)Chains are mixed and disulfide bridges

formYeast cells provide a sterile growth

mediumFinal product is Humulin - chemically

identical to human insulin


Possible Complications of Using Possible Complications of Using Human InsulinHuman Insulinhypoglycemia (low blood sugar) tends to

be more common than with animal insulin


The Role of Restriction The Role of Restriction EndonucleasesEndonucleasesRestriction endonucleases, first

discovered in the late 1960s, are named for preventing invasion by foreign DNA by cutting it into pieces

These enzymes cut at sites within the foreign DNA instead of chewing from the ends

By cutting DNA at specific sites they function as finely honed molecular knives


Naming Restriction EndonucleasesNaming Restriction Endonucleases

Restriction endonucleases are named using the 1st three letters of their name from the Latin name of their source microorganism Hind III◦ First letter is from the genus H from Haemophilus◦ Next two letters are the 1st two letters of the species

name in from influenzae◦ Sometimes the strain designation is included

“d” from strain Rd

◦ If microorganism produces only 1 restriction enzyme, end the name with Roman numeral I Hind I◦ If more than one restriction enzyme is produced, the

others are numbered sequentially II, III, IV, etc.


Restriction Endonuclease SpecificityRestriction Endonuclease Specificity

Restriction endonucleases recognize a specific DNA sequence, cutting ONLY at that sequence◦ These enzymes can recognize

4-bp, 6-bp, 8-bp sequences◦ The frequency of cuts lessens

when the recognition sequence is longer


Restriction Enzyme TerminologyRestriction Enzyme TerminologyA 6-bp cutter will yield DNA fragments

averaging 4000-bp or 4 kilobases (4kb) in length

Heteroschizomers recognize the same DNA sequence but use a different cutting site – they are also called isochizomers

These enzymes cut DNA strands reproducibly in the same place, which is extremely useful in gene analysis


Use of Restriction EndonucleasesUse of Restriction EndonucleasesMany restriction endonucleases make

staggered cuts in the 2 DNA strands◦ This leaves single-stranded overhangs, called

sticky ends that can base-pair together briefly◦ This makes joining 2 different DNA molecules

together much easierStaggered cuts occur when the

recognition sequence usually displays twofold symmetry, palindromes


Restriction-Modification SystemRestriction-Modification SystemWhat prevents these enzymes

from cutting up the host DNA?◦ They are paired with methylases◦ Theses enzymes recognize,

methylate the same siteTogether they are called a

restriction-modification system, R-M system

Methylation protects DNA, after replication the parental strand is already methylated


Basics of type II Restriction EnzymesBasics of type II Restriction Enzymes

No ATP requirement.Recognition sites in double stranded DNA have a 2-fold axis

of symmetry – a “palindrome”.Cleavage can leave staggered or "sticky" ends or can produce

"blunt” ends.


Type II restriction enzyme Type II restriction enzyme nomenclaturenomenclature

EcoRI – Escherichia coli strain R, 1st enzymeBamHI – Bacillus amyloliquefaciens strain H, 1st enzymeDpnI – Diplococcus pneumoniae, 1st enzyme HindIII – Haemophilus influenzae, strain D, 3rd enzymeBglII – Bacillus globigii, 2nd enzymePstI – Providencia stuartii 164, 1st enzymeSau3AI – Staphylococcus aureus strain 3A, 1st enzymeKpnI – Klebsiella pneumoniae, 1st enzyme

02/20/15

Why the funny names?

Asheesh Kumar Pandey ([email protected])

An Experiment Using Restriction An Experiment Using Restriction EndonucleaseEndonuclease

An early experiment used EcoRI to cut 2 plasmids, small circular pieces of DNA independent of the host chromosome

Each plasmid had 1 site for EcoRI◦ Cutting converted circular plasmids

into linear DNA with the same sticky ends◦ The ends base pair

Some ends re-close Others join the 2 pieces

DNA ligase joins 2 pieces with covalent bonds


Plasmids – vehicles for cloningPlasmids – vehicles for cloning

Plasmids are naturally occurring extrachromosomal DNA molecules.

Plasmids are circular, double-stranded DNA.

Plasmids are the means by which antibiotic resistance is often transferred from one bacteria to another.

Plasmids can be cleaved by restriction enzymes, leaving sticky or blunt ends.

Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid.

02/20/15

Tetr

Ampr

OripBR322

4361bp

OripUC18

Ampr

MCS

LacZ


Cloning VectorsCloning Vectors

A cloning vector is a plasmid that can be modified to carry new genes.

Plasmids useful as cloning vectors must have: An origin of replication. A selectable marker (antibiotic

resistance gene, such as ampr and tetr).

Multiple cloning site (MCS) (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers).

Easy to purify away from host DNA.

02/20/15

Tetr

Ampr

OripBR322

4361bp

OripUC18

Ampr

MCS

LacZ

Older cloning vector

Newer cloning vector


Insert – Target DNA

2. Restriction Enzymes

1. PCR product

RE1 RE2RE1

RE1

RE2RE2

T T


VectorsVectorsVectors function as DNA carriers to allow

replication of recombinant DNAsTypical experiment uses 1 vector plus a piece

of foreign DNA ◦ Depends on the vector for its replication◦ Foreign DNA has no origin of replication, the site

where DNA replication beginsThere are 2 major classes of vectors:◦ Plasmids ◦ Phages


Vector requirementsVector requirementsSelectable marker ◦ Resistance to ampicillin, kanamycin, hygromycin, herbicide, etc.◦ Allows only recombinant clones to survive

Multiple cloning site (mcs)◦ many (unique) restriction sites

For vectors that amplify in E. coli cells:◦ Origin of replication (Ori): required for plasmid to replicate in

bacterium◦ Control of copy number (F factor plasmid): reduces copy

number of plasmids in a given cell, lessens problems of rearrangements (chimerism)

For vectors that amplify in yeast cells:◦ Autonomous replication sequence (ARS): similar to Ori◦ CEN: centromere◦ TEL: telomere


Plasmids As VectorsPlasmids As VectorspBR plasmids were developed early but

are rarely used todaypUC series is similar to pBR◦ 40% of the DNA, including tetracycline

resistance has been deleted◦ Cloning sites are clustered together into one

area called the multiple cloning site (MCS)


pBR322 PlasmidpBR322 Plasmid

pBR322 illustrates cloning methods simply◦ Resistance for 2 antibiotics

Tetracycline Ampicillin

◦ Origin of replication between the 2 resistance genes◦ Only 1 site for several

restriction enzymes


pBR322 CloningpBR322 CloningClone a foreign DNA into the PstI site of pBR322 Cut the vector to generate the sticky ends Cut foreign DNA with PstI also – compatible ends Combine vector and foreign DNA with DNA ligase to seal sticky ends Now transform the plasmid into E. coli


Bacterial TransformationBacterial TransformationTraditional method involves incubating

bacterial cells in concentrated calcium salt solution◦ The solution makes the cell membrane leaky,

permeable to the plasmid DNANewer method uses high voltage to drive

the DNA into the cells in process called electroporation


Transformation

Two main methods:

1. Chemical transformation – Chilling cells in the presence of Ca2+ prepares the cell walls to become permeable to plasmid DNA. Cells are briefly heat shocked which causes the DNA to enter the cell

2. Electoporation- making holes in bacterial cells, by briefly shocking them with an electric field of 10-20kV/cm. Plasmid DNA can enter the cell through these holes.

Use of bacterial cells to amplify the DNA of interest


Screening TransformantsScreening Transformants

Transformation produces bacteria with:◦ Religated plasmid◦ Religated insert◦ Recombinants

Identify the recombinants using the antibiotic resistance◦ Grow cells with tetracycline so only cells with plasmid

grow, not foreign DNA only◦ Next, grow copies of the original colonies with ampicillin

which kills cells with plasmid including foreign DNA


Screening With Replica PlatingScreening With Replica PlatingReplica plating transfers clone

copies from original tetracycline plate to a plate containing ampicillin

A sterile velvet transfer tool can be used to transfer copies of the original colonies

Desired colonies are those that do NOT grow on the new ampicillin plate


pUC and pUC and ββ-galactosidase-galactosidaseNewer pUC plasmids have:◦ Ampicillin resistance gene◦ Multiple cloning site inserted into the gene lacZ’

coding for the enzyme β-galactosidase Clones with foreign DNA in the MCS disrupt the ability of

the cells to make β-galactosidase Plate on media with a β-galactosidase indicator (X-gal)

and clones with intact β-galactosidase enzyme will produce blue colonies

Colorless colonies should contain the plasmid with foreign DNA


Lac Z gene

LacZ genepromotor

RNA pol.

Gene expression dogma

DNA

LacZ mRNARibosome

β-galactosidase

RNA

Protein

X-gal BLUE coloniesBLUE colonies

WHITE WHITE coloniescoloniesX-gal


Expression vectors

To yield the product of a cloned gene for further studies

1) Expression vectors with a strong promoter

More mRNA More protein

2) Expression vectors with an inducible promoterForeign proteins when overexpressed could be toxicKeep the gene expression off till it is time to turn it on

a. Drug-inducible (e.g. IPTG)b. Heat-inducible


Expression vectorsTo yield the product of a cloned gene for further studies

1) Expression vectors with a strong promoter

More mRNA More protein

2) Expression vectors with an inducible promoterForeign proteins when overexpressed could be toxicKeep the gene expression off till it is time to turn it on

a. Drug-inducible (e.g. IPTG or arabinose)b. Heat-inducible

3) Expression vectors with a fusion tag for affinity purificationFacilitate the purification of the expressed protein

1) 6 Histidine tag2) Glutathione transferase tag (GST)3) Maltose-binding protein tag


LacZpromotor operator

Repressor

Lac Z gene

LacZpromotor

RNA pol.

RNA pol.

IPTG

IPTG

IPTG

LacZpromotor operator

IPTGIPTG

RNA pol.

IPTG

X-galΒ-galactosidase

X + galactoseCells which produce ß-galactosidase form BLUE coloniesBLUE colonies. Cells without ß-galactosidase production form WHITE WHITE coloniescolonies.


X

XX

X

X

Plasmid without Insert

Plasmid +Insert

without plasmid

Screening

LacZ

pGEM

Insert

WHITE coloniesWHITE colonies BLUE BLUE coloniescolonies

promotor

operator

TT


An Expression Vector for Making a LacZ Fusion Protein

LacZ can be replaced with:

1) Glutathione S-transferase (GST)

2) Maltose-binding protein (MBP)

Affinity Ligand:

Glutathione

Starch (amylose)02/20/15Asheesh Kumar Pandey ([email protected])

A plasmid DNA will be purified from the bacteria cells.

Insert

Vector

Confirmation by digestion with restriction enzyme and separation of the digestion

products on agarose gel

EcoRI

EcoRI

Plasmid DNA will be digested with EcoRI, and analyzed by gel electrophoresis for identification of the clone containing insert.

pGEM


Directional CloningDirectional CloningCut a plasmid with 2 restriction enzymes

from the MCSClone in a piece of foreign DNA with 1

sticky end recognizing each enzymeThe insert DNA is placed into the vector

in only 1 orientationVector religation is also prevented as the

two restriction sites are incompatible


SummarySummaryFirst generation plasmid cloning vectors include

pBR322 and the pUC plasmidspBR322 has ◦ 2 antibiotic resistance genes ◦ Variety of unique restriction sites for inserting foreign DNA◦ Most of these sites interrupt antibiotic resistance, making

screening straightforwardpUC has◦ Ampicillin resistance gene◦ MCS that interrupts a β-galactosidase gene

MCS facilitates directional cloning into 2 different restriction sites


Relaxed, cccDNA(covalently, closed, circular)

Supercoiled DNA

Open (nicked) circular

Gyrase

Topoisomerase

Endonuclease

DNA ligase

Endonuclease

Interconversions of different forms of plasmids

Relaxed Plasmid (multiple copies per cell)

Stringent Plasmid (limited copies per cell)

Non-integrative

Episome (Integrated)

tra genes (conjugative / non-conjugative)


Phages As VectorsPhages As VectorsBacteriophages are natural vectors that

transduce bacterial DNA from one cell to another

Phage vectors infect cells much more efficiently than plasmids transform cells

Clones are not colonies of cells using phage vectors, but rather plaques, a clearing of the bacterial lawn due to phage killing the bacteria in that area


Vectors Host - Features Insert Size

Plasmid E.coli- Introduced by transformation 1-5 kb (electroporation or heat shock)

Phage E.coli- Virus that infects bacteria 10-15 kbIntroduced by transfection

Cosmid E.coli- Plasmid with “cos” sites for 30-45 kb packaging into lambda phage particles. Introduced by infection into E. coli

02/20/15

DNA molecule originating from a virus, a plasmid, or the cell of a higher organism into which another DNA fragment of appropriate size can be integrated without loss of the vector’s capacity for self-replication

Vectors introduce foreign DNA into host cells, where the DNA can be reproduced in large quantities


RNase H

Pol I

RNA II primes DNA synthesis

Replication

RNA II (555 bp)

RNA I (108 bp)

5’5’3’3’

Origin

Infrequently

Frequently

RNA I/ RNA II hybrid + Rop dimer (initial pairing of duplex)

Rop dimer

RNA II inactivated for primer function

No Replication

Plasmid copy number (regulation of replication of Col E1-derived plasmids


Partitioning and segregative stability of plasmids par gene

Incompatibility of plasmidsInability of two different plasmids to coexist in the same cell in the absence of selection pressure (same mechanism of replication control)


λλ Phage VectorsPhage VectorsFirst phage vectors were constructed by

Fred Blattner and colleagues◦ Removed middle region◦ Retained genes needed for phage replication◦ Could replace removed phage genes with

foreign DNAOriginally named Charon phageMore general term, replacement vectors


Phage Vector AdvantagesPhage Vector AdvantagesPhage vectors can receive larger amounts

of foreign DNA◦ Charon 4 can accept up to 20kb of DNA◦ Traditional plasmid vectors take much less

Phage vectors require a minimum size foreign DNA piece (12 kb) inserted to package into a phage particle


Cloning Using a Phage VectorCloning Using a Phage Vector


Genomic LibrariesGenomic LibrariesA genomic library contains clones of all

the genes from a species genomeRestriction fragments of a genome can be

packaged into phage using about 16 – 20 kb per fragment

This fragment size will include the entirety of most eukaryotic genes

Once a library is established, it can be used to search for any gene of interest


Plaque HybridizationPlaque Hybridization

Searching a genomic library requires probe showing which clone contains desired gene

Ideal probe – labeled nucleic acid with sequence matching the gene of interest


CosmidsCosmidsCosmids are designed for cloning large DNA fragments◦ Behave as plasmid and phage◦ Contain

cos sites, cohesive ends of phage DNA that allow the DNA to be packaged into a λ phage head

Plasmid origin of replication permitting replication as plasmid in bacteria

◦ Nearly all λ genome removed so there is room for large inserts (40-50 kb)◦ So little phage DNA can’t replicate, but they are infectious

carrying recombinant DNA into bacterial cells


M13 Phage VectorsM13 Phage VectorsLong, thin, filamentous phage M13Contains:◦ Gene fragment with β-galactosidase◦ Multiple cloning site like the pUC family

Advantage◦ This phage’s genome is single-stranded DNA◦ Fragments cloned into it will be recovered in

single-stranded form


M13 Cloning to Recover Single-M13 Cloning to Recover Single-stranded DNA Productstranded DNA Product

After infecting E. coli cells, single-stranded phage DNA is converted to double-stranded replicative form

Use the replicative form for cloning foreign DNA into MCS

Recombinant DNA infects host cells resulting in single-stranded recombinant DNA

Phage particles, containing single-stranded phage DNA is secreted from transformed cells and can be collected from media


PhagemidsPhagemids

Phagemids are also vectors◦ Like cosmids have aspects of

both phages and plasmids◦ Has a MCS inserted into lacZ’

gene to screen blue staining / white colonies◦ Has origin of replication of

single-stranded phage f1 to permit recovery of single-stranded recombinant DNA◦ MCS has 2 phage RNA

polymerase promoters, 1 on each side of MCS


Bacterial Artificial Chromosomes(BACs) and Bacterial Artificial Chromosomes(BACs) and Yeast Artificial Chromosomes(YACs)Yeast Artificial Chromosomes(YACs)

BACs can hold up to 300 kbs. The F factor of E.coli is capable of

handling large segments of DNA. Recombinant BACs are introduced

into E.coli by electroportation ( a brief high-voltage current). Once in the cell, the rBAC replicates like an F factor.

Example: pBAC108L Has a set of regulatory genes, OriS,

and repE which control F-factor replication, and parA and parB which limit the number of copies to one or two.

A chloramphenicol resistance gene, and a cloning segment.

YACs can hold up to 500 kbs. YACs are designed to replicate as

plasmids in bacteria when no foreign DNA is present. Once a fragment is inserted, YACs are transferred to cells, they then replicate as eukaryotic chromosomes.

YACs contain: a yeast centromere, two yeast telomeres, a bacterial origin of replication, and bacterial selectable markers.

YAC plasmidYeast chromosome DNA is inserted to a unique restriction

site, and cleaves the plasmid with another restriction endonuclease that removes a fragment of DNA and causes the YAC to become linear. Once in the cell, the rYAC replicates as a chromosome, also replicating the foreign DNA.


PROPERTY P1 pBAC pucBAC pCYPAC YAC

Vector Size (kb) 31 6.5 7.2 19.3 11.5

Vector Copy # single single multiple multiple multiple

Insert Size (kb) 75-95 0-300 0-300 0-300 0-2000

Cloning Strategy 2 arms single digest single digest single digest double digest

BamHI/ScaI Bam or Hind Bam or Hind Bam/Sca link Bam/EcoRI

Cloning Method Packaging Electroporate Electroporate Electroporate Spheroplast

Maintenance (copy #) single single single single single

Chimeric Clones (%) 0 2 2 0 (24/24) 20-60

Positive Selection yes no no yes yes

Copy # Induction yes no no yes no

Large Fragment Cloning VectorsLarge Fragment Cloning Vectors


Selected Protein Expression SystemsLiving Hosts:

Escherichia coli

Saccharomyces cerevisiae

Picchia pastoris

Baculovirus

Mammalian tissue culture

Transgenic animals (flies, mice, barnyard animals)

Specialized Systems:

Xenopus oocytes

In vitro transcription-translation

Choosing a System:

-- yield

-- product purification

-- post-translational modifications

-- genetic manipulation of gene/cDNA

-- cost

-- experimental difficulty02/20/15



SummarySummaryTwo kinds of phage are popular cloning vectors

‑ λ phage ‑ Has nonessential genes removed making room for inserts- Cosmids accept DNA up to 50 kb

- M13 phage- Has MCS- Produces single-stranded recombinant DNA

Plasmids called phagemids also produce single-stranded DNA in presence of helper phage

Engineered phage can accommodate inserts up to 20 kb, useful for building genomic libraries


Eukaryotic Vectors and Very Eukaryotic Vectors and Very High Capacity VectorsHigh Capacity Vectors

There are vectors designed for cloning genes into eukaryotic cells

Other vectors are based on the Ti plasmid to carry genes into plant cells

Yeast artificial chromosomes (YAC) and bacterial artificial chromosomes (BAC) are used for cloning huge pieces of DNA


Identifying a Specific Clone With Identifying a Specific Clone With a Specific Probea Specific Probe

Probes are used to identify a desired clone from among the thousands of irrelevant ones

Two types are widely used◦ Polynucleotides also called oligonucleotides ◦ Antibodies


Polynucleotide ProbesPolynucleotide Probes

Looking for a gene you want, might use homologous gene from another organism◦ If already cloned◦ Hope enough sequence similarity to permit

hybridization◦ Need to lower stringency of hybridization

conditions to tolerate some mismatches


Control of Hybridization Control of Hybridization StringencyStringency

Factors that promote separation of two strands in a DNA double helix:◦ High temperature◦ High organic solvent concentration◦ Low salt concentration

Adjust conditions until only perfectly matched DNA strands form a duplex = high stringency

Lowering these conditions lowers stringency until DNA strands with a few mismatches can hybridize


SummarySummarySpecific clones can be identified using

polynucleotide probes binding to the gene itself

Knowing the amino acid sequence of the a gene product permits design of a set of oligonucleotides that encode part of the amino acid sequence

Can be a very quick and accurate means of identifying a particular clone


cDNA CloningcDNA CloningcDNA is the abbreviation for

complementary DNA or copy DNAA cDNA library is a set of clones

representing as many as possible of the mRNAs in a given cell type at a given time◦ Such a library can contain tens of thousands

of different clones


Making a cDNA LibraryMaking a cDNA Library


Reverse Transcriptase PrimerReverse Transcriptase PrimerCentral to successful cloning is the

synthesis of cDNA from an mRNA template using reverse transcriptase (RT), RNA-dependent DNA polymerase◦ RT cannot initiate DNA synthesis without a

primer◦ Use the poly(A) tail at 3’ end of most

eukaryotic mRNA so that oligo(dT) may serve as primer


Ribonuclease HRibonuclease HRT with oligo(dT) primer has made a

single-stranded DNA from mRNANeed to start to remove the mRNAPartially degrade the mRNA using

ribonuclease H (RNase H)◦ Enzyme degrades RNA strand of an RNA-DNA

hybrid◦ Remaining RNA fragments serve as primers

for “second strand” DNA using nick translation


Nick TranslationNick TranslationThe nick translation process

simultaneously:◦ Removes DNA ahead of a nick◦ Synthesizes DNA behind nick◦ Net result moves or translates the

nick in the 5’ to 3’ directionEnzyme often used is E. coli

DNA polymerase I◦ Has a 5’ to 3’ exonuclease activity ◦ Allows enzyme to degrade DNA

ahead of the nick


Trailing Terminal TransferaseTrailing Terminal Transferase

Don’t have the sticky ends of genomic DNA cleaved with restriction enzymes

Blunt ends will ligate, but inefficientGenerate sticky ends using terminal

deoxynucleotidyl transferase (TdT), terminal transferase with one dNTP◦ If use dCTP with the enzyme◦ dCMPs are added one at a time to 3’ ends of the cDNA◦ Same technique adds oligo(dG) ends to vector◦ Generate ligation product ready for transformation


Vector ChoiceVector ChoiceChoice based on method used to detect

positive clonesPlasmid or phagemid like pUC or pBS will

be used with colony hybridization and a labeled DNA probe

If λ phage like λgt11, cloned cDNA under control of lac promoter for transcription and translation of the cloned gene and antibody screening


Rapid Amplification of cDNA Rapid Amplification of cDNA EndsEndsIf generated cDNA is not full-length,

missing pieces can be filled in using rapid amplification of cDNA ends (RACE)

Technique can be used to fill in either the missing portion at the 5’-end (usual problem)

Analogous technique can be used to fill in a missing 3’-end


5’-RACE5’-RACEUse RNA prep containing

mRNA of interest and the partial cDNA

Anneal mRNA with the incomplete cDNA

Reverse transcriptase will copy rest of the mRNA

Tail the completed cDNA with terminal transferase using oligo(dC)

Second strand synthesis primed with oligo(dG)


SummarySummaryMake cDNA library with synthesis of cDNAs one strand at

a time◦ Use mRNAs from a cell as templates for 1st strands, then 1st

strand as template for 2nd

◦ Reverse transcriptase generates 1st strand◦ DNA polymerase I generates the second strands

Give cDNAs oligonucleotide tails that base-pair with complementary tails on a cloning vector

Use these recombinant DNAs to transform bacteriaDetect clones with:◦ Colony hybridization using labeled probes◦ Antibodies if gene product translated

Incomplete cDNA can be filled in with 5’- or 3’-RACE


Methods of Expressing Cloned Methods of Expressing Cloned GenesGenesCloning a gene permits Production of large quantities of a

particular DNA sequence for detailed study

Large quantities of the gene’s product can also be obtained for further use◦ Study ◦ Commerce


Expression VectorsExpression Vectors

Vectors discussed so far are used to first put a foreign DNA into a bacterium to replicate and screen

Expression vectors are those that can yield protein products of the cloned genes◦ For high level expression of a cloned gene best

results often with specialized expression vectors◦ Bacterial vectors have a strong promoter and a

ribosome binding site near ATG codon


Fusion ProteinsFusion Proteins

Some cloning vectors, pUC and pBS, can work as expression vectors using lac promoter

If inserted DNA is in the same reading frame as interrupted gene, a fusion protein results◦ These have a partial β-

galactosidase sequence at amino end◦ Inserted cDNA protein

sequence at carboxyl end02/20/15


Inducible Expression VectorsInducible Expression VectorsMain function of expression vector is to yield the

product of a gene – usually more is betterFor this reason, expression vectors have very strong

promotersPrefer keep a cloned gene repressed until time to

express◦ Large quantities of eukaryotic protein in bacteria are

usually toxic◦ Can accumulate to levels that interfere with bacterial

growth◦ Expressed protein may form insoluble aggregates, inclusion

bodies


Controlling theControlling the lac lac Promoter Promoterlac promoter is somewhat inducible◦ Stays off until stimulated◦ Actually repression is incomplete or leaky◦ Some expression will still occur

To avoid this problem, express using a plasmid or phagemid carrying its own lacI repressor gene, such as pBS


Arabinose PromoterArabinose PromoterThe hybrid trc promoter combines

strength of the trp (tryptophan operon) promoter with inducibility of lac promoter

Promoter from ara operon, PBAD, allow fine control of transcription◦ Inducible by arabinose, a sugar◦ Transcription rate varies with arabinose

concentration


Tightly Controlled PromoterTightly Controlled Promoter

Lambda (λ) phage promoter, PL, is tightly controlled

Expression vectors with this promoter-operator system are used in host cells with temperature-sensitive λ repressor gene◦ Repressor functions are low temperatures◦ Raise temperature to nonpermissive

temperature, the repressor doesn’t function and cloned gene is expressed



cI repressor

Bacterial chromosome

Bacterial chromosome

Po Ptrp λcI repressor

Transcription

No Transcription

Po PL ATG Gene of Interest

Vector

Vector

Tryptophan

trp repressor

No Transcription

λcI repressor

Transcription

ATG Gene of Interest

Po

Po

Ptrp

PL

+ Tryptophan

- TryptophanRegulation of gene expression


SummarySummaryExpression vectors are designed to yield

the protein product of a cloned geneWhen a lac inducer is added, cell begins

to make T7 polymerase which transcribes the gene of interest

Many molecules of T7 polymerase are made, so gene is turned on to a very high level with abundant amount of protein product made


Expression Vectors That Produce Expression Vectors That Produce Fusion ProteinsFusion Proteins

Most vectors express fusion proteins◦ The actual natural product of the gene isn’t made◦ Extra amino acids help in purifying the protein product

Oligohistidine expression vector has a short sequence just upstream of MCS encoding 6 His◦ Oligohistidine has a high affinity for divalent metal ions like

Ni2+ ◦ Permits purification by nickel affinity chromatography◦ His tag can be removed using enzyme enterokinase without

damage to the protein product


Oligohistidine Expression VectorOligohistidine Expression Vector


Fusion Proteins in Fusion Proteins in λλgt11gt11

This phage contains lac control region and lacZ gene

Products of gene correctly inserted will be fusion proteins with a β-galactosidase leader


Antibody Screening With Antibody Screening With λλgt11gt11Lambda phages with cDNA

inserts are platedProtein released are

blotted onto a supportProbe with antibody to

proteinAntibody bound to protein

from plaque is detected with labeled protein A

Partial cDNAs can be completed with RACE


SummarySummaryExpression vectors frequently produce fusion

proteins◦ One part of the protein comes from coding

sequences in the vector◦ Other part from sequences in the cloned gene

Many fusion proteins have advantage of being simple to isolate by affinity chromatography

Vector lgt11 produces fusion proteins that can be detected in plaques with a specific antiserum


Bacterial Expression System Bacterial Expression System ShortcomingsShortcomingsThere are problems with expression of

eukaryotic proteins in a bacterial system◦ Bacteria may recognize the proteins as foreign and

destroy them◦ Posttranslational modifications are different in

bacteria◦ Bacterial environment may not permit correct

protein foldingVery high levels of cloned eukaryotic proteins

can be expressed in useless, insoluble form


Eukaryotic Expression SystemsEukaryotic Expression SystemsAvoid bacterial expression problems by

expressing the protein in eukaryotic cellInitial cloning done in E. coli using a shuttle

vector, able to replicate in both bacterial and eukaryotic cells

Yeast is suited for this purpose◦ Rapid growth and ease of culture◦ Still a eukaryote with more appropriate

posttranslational modification◦ Secretes protein in growth medium so easy

purification02/20/15


Use of Baculovirus As Expression Use of Baculovirus As Expression VectorVector

Viruses in this class have a large circular DNA genome, 130 kb

Major viral structural protein is made in huge amounts in infected cells◦ Promoter for this protein, polyhedrin, is very active◦ These vectors can produce up to 0.5 g of protein

per liter of medium◦ Nonrecombinant viral DNA entering cells cannot

result in infectious virus as it lacks an essential gene supplied by the vector


Animal Cell TransfectionAnimal Cell TransfectionCalcium phosphate◦ Mix cells with DNA in a phosphate buffer◦ Then solution of calcium salt added to form a

precipitate◦ Cells take up the calcium phosphate crystals which

include some DNALiposomes◦ DNA mixed with lipid to form liposomes, small vesicles

with some of the DNA inside◦ DNA-bearing liposomes fuse with cell membrane

carrying DNA inside the cell


Choosing a Mammalian Cell Expression SystemAdvantages:

Cell biology (ie. Localization, cell cycle etc.)

Post-translational modification (mammalian)

Improving expression vectors and transfection methods

Disadvantages:

Low yield of expressed proteins

Relatively expensive (tissue culture costs)

Must use shuttle vectors

Key terms:

Transient transfection: introduction of episomal expression vector into mammalian tissue culture cells for short term expression experiments.

Stable transfection: introduction and integration of vector into the host cell chromosome for long-term expression studies.





SummarySummary

Foreign genes can be expressed in eukaryotic cells

These eukaryotic systems have advantages over prokaryotic ones◦ Made in eukaryotic cells tend to fold properly and

are then soluble rather than aggregated into insoluble inclusion bodies◦ Posttranslational modifications are made in a

eukaryotic manner


Using the Ti Plasmid to Transfer Using the Ti Plasmid to Transfer Genes to PlantsGenes to Plants

Genes can be introduced into plants with vectors that can replicate in plant cells

Common bacterial vector promoters and replication origins are not recognized by plant cells

Plasmids are used containing T-DNA◦ T-DNA is derived from a plasmid known as tumor-

inducing (Ti)◦ Ti plasmid comes from bacteria that cause plant

tumors called crown galls


Ti Plasmid InfectionTi Plasmid InfectionBacterium infects plant, transfers Ti

plasmid to host cellsT-DNA integrates into the plant DNA

causing abnormal proliferation of plant cells

T-DNA genes direct the synthesis of unusual organic acids, opines which can serve as an energy source to the infecting bacteria but are useless to the plant


Ti Plasmid Transfers Crown GallTi Plasmid Transfers Crown Gall


Use of the T-DNA PlasmidUse of the T-DNA Plasmid


Gene Cloning

Education

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copies of insulin gene

human insulin

cloned gene

coli bgalactosidase

impaired production

use of recombinant dna

dna techniqueinsulin